1 INTRODUCTION

Natural rubber (NR) is an important high-performance material, which has incomparable advantages over petroleum-derived synthetic rubbers in many applications requiring abrasion, heat dispersion, resilience, and other desirable properties (van Beilen & Poirier, 2007). As a valuable biopolymer, NR can be used to manufacture many rubber products, including latex gloves and tires (Cherian et al., 2019). To date, the production of NR in the world mainly comes from the Brazilian rubber tree Hevea brasiliensis (Cornish, 2017), and NR is also faced with fluctuating prices and increasing demand with the rapid economic development in many countries (van Beilen & Poirier, 2007). Therefore, we need to find some renewable NR materials to replace petroleum-derived products. Russian dandelion Taraxacum kok-saghyz Rodin (TKS) is considered to be a promising renewable NR material to replace petroleum-derived products (Clement-Demange et al., 2007). It is native to Xinjiang areas in China and Kazakhstan, and often grows in salinized meadows, flood plain meadows, and farmland canals (Krotkov, 1945; Whaley & Bowen, 1947). After recognizing the importance and urgency of developing alternative sources for NR, many countries have collected a large number of TKS germplasm resources and strengthened the basic research on TKS (Luo et al., 2017). For example, Lin et al. (2018) had sequenced the reference genome of TKS. Inulin content, rubber content, and root biomass of TKS population were also evaluated based on agronomic characteristics and molecular markers (Arias et al., 2016). Moreover, genetic resources play an important role in germplasm identification, breeding strategy and crop improvement. Previous studies about the population diversity of TKS have been conducted using eSSRs and gSSRs as markers, such as McAssey et al. (2016) running rudimentary species diversity study of TKS using eSSRs and Nowicki et al. (2019) providing many additional insights using gSSRs on the USDA germplasm of TKS. The evaluation of the genetic diversity and evolutionary patterns in TKS is extremely important for the constitution of repository fields, obtained from the selection of genotypes collected from wild plants; the genetic diversity needs to be assessed and eventually integrated; finally, a rational conservation program for TKS germplasm resources needs to be associated with a genetic diversity evaluation.

High-throughput molecular markers, such as amplified fragment length polymorphism and random amplified polymorphic DNA, are effective in elucidating and identifying genetic backgrounds (Abdollahi et al., 2015; Bhagyawant, 2016; Cheng et al., 2015; Fu et al., 2015). These marker systems are economical, simple, and automated compared with whole-genome resequencing, but not numerous enough to saturate large populations. With the rapid development of deep sequencing technologies, the aforementioned shortcomings of these traditional markers are being resolved. These emerging sequencing technologies make it possible for high-throughput identification of SNPs in many species, including those without reference genomes (Rimbert et al., 2018; Zhou et al., 2017). For example, SNPs, an emerging molecular marker type, are efficient and powerful for population genetics studies. These approaches have facilitated the whole-genome resequencing for several hundred lines, marker genotyping platforms, the development of high-density genetic maps, and markers related to agronomic traits (Varshney et al., 2019). Nowadays, we can identify causal genetic features for breeders to perform biological interventions by integrating genotype and phenotype data effectively (Ramstein et al., 2018; Wallace et al., 2018; Zhang et al., 2018), such as genome-wide association study (GWAS). Moreover, high-throughput SNP molecular markers have not been used to evaluate the genetic diversity and evolutionary patterns of TKS germplasm resources worldwide so far (Cherian et al., 2019).

In this study, a population evolutionary analysis of 58 TKS individuals collected from different distribution regions was implemented to (a) characterize TKS population genetic structure and (b) identify candidate genes under natural selection for TKS germplasm adaptation to the Yili River Valley based on SNP markers developed using Illumina GBS approach.

2 MATERIALS AND METHODS

3 RESULTS

3.1 Agronomic traits diversity and cluster analysis

The ANOVA results of eight agronomic traits showed that 58 TKS individuals were significantly different in terms of their agronomic traits. The highest range of phenotypic variance (60.37) was observed in BF, whereas the least value (0.21) was attributed to LT (Table 1, Table S1). The highest phenotypic coefficient of variation (42%) was denoted to SN (Table 1, Table S1). Scape yield of per plant ranged from 3 to 30 (Table 1, Table S1). The high values of CD and LL were in TS location, and the high BF value was found in Kazakhstan. Locations TS and Kazakhstan also showed the highest LW and LT. For SN, SL, and SD, the values were evenly distributed among the individuals in different locations (Table 1). Clustering of 58 TKS individuals based on their agronomic traits revealed that they were divided into three groups (Figure 1a). First group (I) were exclusively Kazakhstan population (D) individuals, nine out of 58 individuals studied here. Second group (II) possessed about 10% of all individuals, only including six individuals in population C. Third group (III) made 75% of the individuals, from all geographical regions, including populations A, B, C, and D (Figure 1a). Based on their values for CD, BF, LL, LW, and LT among the 58 evaluated individuals, populations C and D were assigned to separate clusters, respectively.

TABLE 1. Statistics analysis for agronomic traits of TKS in the study

Traits	Mean ± SD2	Range	Variance	Skew	Kurtosis	CV (%)
CD (cm)	18.59 ± 5.97	7.41–35.39	35.59	0.85	1.05	32
BF (day)	332.86 ± 7.77	325–355	60.37	1.54	1.35	2
LL (cm)	8.96 ± 3.18	3.66–16.54	10.14	0.58	−0.45	36
LW (cm)	2.09 ± 0.62	0.90–4.10	0.39	1.13	2.26	30
LT (cm)	1.54 ± 0.46	0.76–2.79	0.21	1.07	0.82	30
SN	13.10 ± 5.51	3–30	30.34	0.67	0.80	42
SL (cm)	14.67 ± 4.77	5.31–28.80	22.75	0.14	0.31	33
SD1 (cm)	2.02 ± 0.58	0.80–3.19	0.33	0.08	−0.41	29

• Abbreviations: BF, beginning of flowering; CD, crown diameter; CV, coefficient of variance; LL, leaf length; LT, leaf thickness; LW, leaf width; SD¹, scape diameter; SD², standard deviation; SL, scape length; SN, scape number.

3.3 Population diversification in TKS germplasms

AMOVA analysis showed that the variation distributed within TKS populations (53.30%) and among TKS populations (46.70%) were similar (Table S4), whereas significant differentiation (F_ST > 0.15) existed between different TKS individuals. For instance, A and B populations were significantly different from C population. D population was significantly different from A and C populations, and still be significantly different with B population (Table S5). To further clarify the genetic relationships among 58 TKS individuals, high-quality SNPs were used to investigate phylogenetic relationships. The cross-validation error of K values setting from 2 to 10 indicated that the highest peak occurred at K = 3 and it was considered as reasonable modelling choice. Combined with the neighbor-joining (NJ) tree (Figure 1b), population structure (Figure 2a), and principal component analysis (Figure 2b), these TKS individuals could be divided into three groups: group I contained the majority of individuals from C population (12, 85.71%); group II included the majority of individuals from D population (9, 64.29%) and two individuals in B population; and group III contained all individuals in A population and most individuals in B population (13, 86.67%), as well as a few individuals in C and D populations (Figures 1b and 2a,b). These findings are consistent with the results of population genetic diversity analysis based on Tajima's D value, which indicated population sizes in A and B locations expanded, whereas population sizes in C and D locations shrank suddenly. Moreover, significant gene introgression from A and B populations to C population, C population to A population and D population to B population (Figure 2c) were detected by TreeMix model. Phylogenetic analysis found that 58 wild TKS individuals have a complex genetic evolution relationship (Figure 1b). Twelve individuals in C population formed a single clade, nine individuals in D population and two individuals in B population formed a single clade, whereas the remaining individuals were clustered into another group with an admixture. In addition, population structure and PCA analysis were carried out and results evidenced a strong genetic differentiation of TKS in Kazakhstan and Xinjiang area (Figures 1b and 2a,b).

3.4 Candidate genes of natural selection regions during TKS evolution

To identify potential selective sweep during wild TKS evolutionary process in geographically distant Kazakhstan and Xinjiang, the distribution of p and F_ST were used and the genome regions with extremely low or high p ratio and high F_ST value (top 5% θπ-sample/θπ-control ≥ 2.2318 or θπ-sample/θπ-control ≤ 0.3733 and F_ST ≥ 0.1916) were considered as strong selective sweep. These results suggested that genome regions in AB population affected by natural selection have a lower level of polymorphism (median θπ-sample/θπ-control = 0.89) compared with CD populations. There are many genomic regions with strong selective sweep signals in AB population (0.40% of the genome and containing 80 genes) compared with CD populations (0.02% of the genome and containing 0 genes) (Figure 3a), which reflects a relatively higher inbreeding under natural selection and thus fewer recombination events and skewed allele frequency spectra in AB population compared with CD populations. To further understand the potential functions of these genes in selective sweep regions of AB population, GO and KEGG pathway enrichment analysis were implemented. GO analysis showed that most candidate genes were mainly enriched in these GO terms such as catalytic activity, transporter activity, structural molecule activity, and molecular function regulator (Figure 3b). KEGG enrichment analysis indicated that the top two enriched pathways were plant hormone signal transduction and limonene degradation (Figure 4; Table S6).

4 DISCUSSION

Diversity evaluation of TKS germplasm in different regions provides important breeding information for the utilization of genetic diversity. However, compared with other crops, the population genomic research of TKS has been largely limited due to lack of applicable abundant molecular markers. The combination of SNP molecular markers and agronomic traits for evaluating the genetic diversity of species is helpful to precisely analyze genetic diversity and population structure within species (Ambreen et al., 2018; Arzani & Ashraf, 2016). This method has been widely used in genetic diversity analysis in various crops such as safflower (Golkar et al., 2011), Nigella sativa L. (Golkara & Nourbakhshb, 2019), and Triticum urartur (Wang et al., 2017), whereas the genetic diversity of TKS has not been studied by this method nowadays. Therefore, 58 TKS individuals were collected in this study and divided into three different groups based on SNP molecular markers and agronomic traits. 80 candidate genes under natural selection for TKS germplasm adaptation to the Yili River Valley were further identified.

Based on the agricultural morphological data, there is a logical similarity between the TKS individuals assigned to the same group and their geographic locations, although a few individuals in C and D population were joined into population AB. It was consistent with the results of phylogenetic tree and principal component analysis based on SNPs molecular marker. And gene introgression from C population to A population and D population to B population also occurred in this study. Hence, we speculate the phenomenon could be due to the influence of various factors, such as stable genetic mutations, substitution or mixture of germplasm across the areas that migrate over long distances, inter-regional plant material exchange, gene flow, climate adaptation and environmental influences on genetic variation (Ramanatha & Hodgkin, 2002) or the profound influence of environmental factors, similar to reports of wheat (Najaphy et al., 2012) and safflower (Golkar et al., 2011). Previous research found that common origin, convergent evolution, and subsequent natural selection may result in accessions from separate regions clustered in a common group (Reeves et al., 2012). Additionally, mutations, recombination, the number of active alleles, genetic drift, and genetic structure also could influence the amount of variation within a population (Ambreen et al., 2018). It is important to obtain a lot of individuals representing the highest possible genetic distance from the entire TKS collections (e.g., Kazakhstan genotypes and Tekes genotype in Xinjiang), which would contribute to their extraction of genes useful for breeding and exploitation of genetic resources. Not surprisingly, the highest similarity between TKS individuals in Kazakhstan and Tekes County in Xinjiang was closely dependent on the smallest genetic distance.

Population structure analysis suggested our entire TKS collection could be clustered into three groups. A few individuals from Kazakhstan and Xinjiang Tekes, as well as all individuals in population A and most in population B, were considered as one group, which is consistent with the results of other clustering methods mentioned above. The result revealed a difference in the genetic structure of TKS individuals grown in Kazakhstan and Tekes County in Xinjiang compared with the other individuals grown in Xinjiang Zhaosu County. Meanwhile, genetic admixture was also found in accessions from populations C and D to population AB, which consistent with the flow of the Tekes River from west to east. Population genetic diversity analysis indicated that population AB expanded, whereas populations C and D shrank. The frequent human activities and the destruction of the wild vegetation in there may lead to the decrease in the genetic diversity of TKS populations in Zhaosu County of Xinjiang. Recently, the ecological environment of Zhaosu County has improved (Ahan et al., 2017) and the population may have expanded after the temporary bottleneck. The admixture among individuals was also reported in other crops such as Nigella sativa L. (Golkara & Nourbakhshb, 2019), safflower (Ambreen et al., 2018), and Simrouba glauca (Kumar & Agrawal, 2017). Comparing cluster results based on agronomic traits with those based on SNPs, it shows that they were almost the same. However, the limited exposure of SNPs to artificial selection, and the coverage of SNP molecular markers to coding and noncoding genomic regions, could sometimes lead to a lack of similarity between molecular markers and morphological variations. Moreover, the top two KEGG-enriched pathways of candidate genes in genomic regions with strong selective sweep signals in AB population were plant hormone signal transduction and limonene degradation. Previous studies have shown that there is a signal transduction network between plant hormone regulation and plant environmental adaptability, that is, plant hormone interactions can regulate plant adaptation to the environment (such as resistance to freezing, high temperature, salt, etc.) (Hu et al., 2017; Kurowska et al., 2020; Wingler et al., 2020). Therefore, the genetic diversity and selection pressures may enable the TKS to adapt to a variety of environmental conditions in different geographical regions.

5 CONCLUSIONS AND OUTLOOK

The results in this study revealed the potentials of SNP markers in evolutionary studies, including genotype distinctness and population genetics. Meanwhile, it points out the scope and direction of further TKS research, such as genome-wide association study of rubber contents traits based on high-throughput sequencing by using a wide range of accessions from each geographical location. The good varieties of TKS individuals could improve the rubber production capacity worldwide. With the increasing demand for NR and limitations of H. brasiliensis production systems, genetic engineering approaches to generate NR-enriched genotypes of alternative NR plants based on the identification of rubber candidate genes are of great importance.

CONFLICT OF INTEREST

None of the authors have conflicts of interest.

AUTHOR CONTRIBUTION

Yan Zhang: Data curation (lead); Formal analysis (equal); Writing-original draft (lead). Hailong Ren: Funding acquisition (equal); Resources (equal); Software (equal). Xuechao Zhang: Formal analysis (equal); Writing-original draft (equal). Li Wang: Data curation (equal). Qiang Gao: Resources (equal). Abudukeyoumu Abudurezike: Visualization (equal). Qingqing Yan: Software (equal). Zifeng Lu: Data curation (equal). Yonggang Wang: Investigation (equal). Qiuhai Nie: Investigation (equal). Lin Xu: Funding acquisition (lead); Project administration (lead); Writing-review & editing (equal). Zhibin Zhang: Formal analysis (lead); Project administration (equal); Software (equal); Writing-review & editing (lead).